DE1645979C3 - Process for the preparation of ribonucleoside-5'-monophosphate - Google Patents

Description

The invention relates to a method for preparing ribonucleoside S'-monophosphates of general formula

HO O
Ν B. \ Il
P- \
/ HO DH -Ο — CH ₂ O / ^x OH C

in which B denotes the remainder of a purine or a pyrimidine base, by selective phosphorylation of the 5'-hydroxyl group of the corresponding ribonucleoside which is unprotected in the 2 'and 3' positions.

Ir

Attempts have already become known to phosphorylate unprotected nucleoside; for example Gulland and Hobday have the reaction of guanosine with phosphorus oxychloride in pyridine investigated (see Journal of Chemical Society, 1940, pp. 746 to 752); Barker and Foil did similar studies on adenosine (see Journal of Chemical Society 1957, pp. 3798 to 3800).

ro The fact that the phosphorylation of unprotected nucleoside is neither industrial nor on a laboratory scale is applied, should be due to the following two reasons: Under Under the known reaction conditions, unprotected nucleoside is less of a phosphorylation reaction accessible as a protected nucleoside; the conversion rate of the reaction is slow; in addition, the phosphorylation of unprotected nucleoside leads to, among other things, 2'- and 3'-monophosphates, 3 '(or 2'), 5'-dipho5phate and dinucleoside phosphate as by-products instead of too the desired 5'-monophosphate and thus has only a low selectivity.

In order to obtain the desired 5'-monophosphate, a method has heretofore been used that is can be represented by the following chemical equation:

in which B has the meaning given, in a reaction mixture containing 0.2 to 3 1 of nitromethane, nitroethane, tetrahydrofuran, dioxane, acetonitrile, propionitrile or dichloromethane, 2 to 12 moles of phosphorus oxychloride, 2 to 6 moles of water and 1 mole of the ribonucleoside Contains 1.5 to 10 mol of pyridine, picoline, lutidine or collidine, is reacted at a reaction ^{temperature of -30 to +30 0} C, the reaction mixture is thoroughly mixed with water and the 5'-monophosphate is isolated after the hydrolysis reaction has ended. 2. The method according to claim 1, characterized in that less than the equimolar amount of water, based on the phosphorus oxychloride, is used.

HIGH ₂ OB
\

OH OH
UP, OB

O O

(Π)

R R

HO O

POCH ₂ OB
HO

O O

60

(HI)

RR
HO O

PO-CH ₂ OB
HO

(IV)

OH OH

1 64S979

where B is a purine or a pyrimidine base and R denotes an alkyl, an aryl group or a hydrogen atom.

In the reactions explained above, reaction II corresponds to an addition of protective Groups, e.g. B. of acetone to the hydroxyl groups in the 2'- or ^ '- position, while the reaction III the phosphorylation of the 5'-hydroxyl group and reaction IV the hydrolysis of the protective groups represents.

Examples of these known processes in which the T and 3′-hydroxyl groups are protected before the phosphorylation of the 5'-hydroxyl group in rjbonucleosides are given in French patents 1434511 and 14.18186. In these known processes, the nucleoside is used for protection in one case the secondary hydroxyl groups are reacted with acetone, in the other case with complex formation with metaboric acid. In a further stage , the S'-hydroxyl group is first phosphorylated and then the protective groups are split off.

In contrast, the invention is based on the object of a method for producing RmonucIeosid-5'-monophosphates the more general. formula

HO O

P-O-CH, 0

HO

OH UH

in the B the remainder of a purine or a pyrimidine base means by selective phosphorylation of the 5'-hydroxyl group of the corresponding in 2'- and 3'-position to make unprotected ribonucleoside accessible in a simpler and more economical way Manner can be carried out and leads to high yields of the ribonucleoside S'-monophosphate. The inventive method is characterized in that a ribonucleoside of the general formula

HO-CH ₂ OB

H [IH

OH OH

in which B has the meaning given. in a reaction mixture based on 0.2 to 31 mol of the ribonucleoside nitromethane. Nitroethane. Tetrahydrofuran, dioxane, acetonitrile. Propionitrile or dichloromethane, 2 to 12 moles of phosphorus oxychloride. 2 to 6 moles of water and 1.5 to 10 moles of pyridine, picoline, lutidine or collidine, reacted at a reaction temperature of -30 to +30 ⁰ C, the reaction mixture thoroughly mixed with water and after the hydrolysis reaction the 5 ' -Mono phosphate isolated.

It is obvious that the known processes in which protected ribonucleosides are used, even if one works without isolating the intermediate products, are more complicated than the selective phosphorylation according to the invention, since the reactions II and IV shown in the formula scheme above have to be carried out .

On the other hand, according to the cited method by Barker and Foil, 5'-, 3'- and 2'-monophosphates are obtained in a ratio of 6: 3: 1, and the yield of 5'-monophosphate is only 6%, based on the starting nucleoside. In contrast

ίο amounts to in the method according to the invention Conversion rate of the nucleoside almost 100% and the selectivity of 5'-phosphorylation always exceeds 90%. The method according to the invention can be explained in detail as follows.

First of all, amounts of one of the specified polar organic solvents, namely nitromethane, nitroethane, tetrahydrofuran, dioxane, acetonitrile, propionitrile or dichloromethane, within the specified limit values are used. Phosphorus oxychloride . Water and one of the specified cyclic tertiary amines, namely pyridine. Piccoline. Lutidine or collidine. mixed with one another: to the resulting mixture, which is kept at a temperature within the specified range, the ribonucleoside is added with stirring.

The reaction mixture, which initially forms a suspension, gradually goes as the reaction proceeds into a clear solution. The resulting reaction mixture becomes cold in a large amount

} o Water or poured onto ice water to obtain an aqueous solution. The reaction mixture can then be treated with the usual methods for separating and purifying nucleosides, e.g. B. using animal charcoal and ion exchange resins. whereby ribonucleoside 5'-monophosphate is obtained.

When performing the method according to the invention are the mixing ratio of the components of the reaction mixture and the selection of the The above-mentioned organic solvents and tertiary amines are extremely important.

The polar organic solvents to be used in the process of the present invention are Solvent with a high dielectric constant. Among the specified substances will be Nitromethane and acetonitrile in particular; preferred.

The amount of these solvents in the process according to the invention depends on the type of solvent and the starting nucleoside and also on the amounts of phosphorus oxychloride, organic base and water a'o. The amounts to be used are in the range from 0.2 to 3 liters per mole of starting nucleoside. The use of these solvents in an amount of less than 0.2 l per mole of nucleoside causes inconvenient working conditions because of the high viscosity of the reaction mixture, while if more than 3 1 per mole of nucleoside is used, the amount of phosphorus oxychloride and organic base would have to be increased, which is economical Viewpoint is undesirable. These organic solvents can be used singly or as a mixture of two or more solvents . The amount of phosphorus oxychloride to be used in the process according to the invention is closely related to the type and amount of the organic solvent and the amounts of cyclic tertiary amine and water. The amount of phosphorus oxychloride to be used is 2 to

5 ^T 6

12 moles per mole of starting nucleoside. The use is in the range from -30 to +30 "C. At higher temperatures of phosphorus oxychloride in lower amounts, a decomposition reaction that is too strong than 2 mol per mol of nucleoside results in a very low one, which makes the process uninteresting Reaction, while other- at lower temperatures the reaction rate is very low using more than 12 moles for a 5 'rate and too long a reaction slowdown in the conversion rate is required. The cyclic tertiary amines used in the gene according to the invention is preferably 30 minutes to 10 hours,
have a strong influence on the rate of urination, although the reaction temperature and duration have one effect and the selectivity of the reaction. Pyridine, picoline, lutidine, mixtures and the selectivity of 5'-phosphoryl collidine were found to have a certain influence on the appearance of the reaction tertiary amines. Having the use of primary, lation, they are not of so great importance.
It is not advisable to use the method according to the invention. The following examples serve to illustrate the. Invention.

The specified cyclic tertiary amines are bis 1 1
in an amount of 1.5 to 10 moles per mole

Nucleoside used. A smaller excess of To a mixed solution containing 20 ml of aceto-

cyclic tertiary amine via an equimolar 20 nitrile, H ml (0.087 mol) phosphorus oxychloride. 1 ml

Amount per mole of starting nuclurfactant leads to usual (0.056 mole) water and 7.9 m '(0.098 mole) pyridine.

Hch to a moderately satisfactory result. that was kept at 0 to 20 ° C, was 5.4 g

The use of less than an equimolar (0.022 mole) inosine is given with stirring. To

Amount Leads to a noticeable delay in the 4 hour stirring, the reaction mixture was in

Reaction rate and reduction in output 25 poured about 50 ml of cold water and stirring

Yield of 5'-nucleotide, while an amount of more than sth> 1 hour at 0 to 5 ^C C continued.

10 moles not only from an economic point of view. The resulting aqueous solution was treated with a

from is desired, but also added the selectivity of the aqueous solution of sodium hydroxide.

5'-phosphorylation adversely affected. The cycli- um increases the pH of the resulting mixture

see tertiary amines are set individually or as mi- 30 about 1; it contained 99% phosphate

used. bonds and 96% inosine 5'-monophosphate. then

In the process according to the invention, the mixture was left to run through a column, The amount of water used is closely related to the 50 g of activated charcoal contained, with inosinic acid adsorder Amount of phosphorus oxychloride and organic beer. After the column with aqueous Base; ausbeiden within the stated range of 0.1 η hydrochloric acid and a small amount of water Ingredients, the amount of water is 2 until washed. the content was marked with a solution 6 Moles per mole of starting nucleoside. If more than mixture of 3% ammonia and 10% methanol in 6 moles of water are used, the un- water is eluted and the resulting eluate is cursed Supported side reactions and dried the reduced pressure. The residue was Amount of purine base and other by-products 40 dissolved in a small amount of water, the solution elevated. If the amount of water was unusually small, it was sent through a column that was about 20 ml is in comparison to the optimal amount so that an ion exchanger in the Η-form runs, and Process not always reproducible, and immediately - the column was washed with water. The timely the yield of 5 'nucleotide is reduced. flowing liquid and the washing liquids were inside the stated range of quantities is 45 denier with an aqueous solution of sodium hydroxide less water than the equimolar amount. adjusted to a pH of 7.7. The mixture based on phosphorus oxychloride. preferable. became a syrup under reduced pressure

There is no particular restriction on looking-focused. After the concentrated solution

the way in which water is added. solution left in an ice container overnight,

however, addition methods are an instant 50 detached crystals, which are filtered off and at 11OC

Reaction of water with phosphorus oxychloride were dried under reduced pressure; man

Causes, e.g. B. the addition of a mixture of ter- received 7.1 g of Dinptriuminosin-5'-monophosphate in

tertiary cyclic amine and water to phosphorus 98% purity. Furthermore, by adding

oxychloride, not advisable. Alcohol and let the mixture stand a little

The reaction temperature and time are also obtained from the filtrate.

important conditions for the reaction if free- _R. 17th

end results are to be achieved. At the ρ e 1 s ρ 1 e I

The method according to the invention is similar to that in Example 1 was repeated with the difference

other former reactions the reaction increase that 10 ml (0.102 mol) «picoline instead of

speed increased with increasing temperature and 60 pyridine were used. 7.2 g of Na-

decreased with falling temperature. In the case of phosphorus inosinate in 95% purity,

phorylation of nucleoside with phosphorus oxychloride _R. ■ 1 -s

one should take into account that when passing the example

the optimal 1 temperature the decomposition of the mixture to a mixed solution, consisting of 20 ml

The initial nucleoside of the nucleotide formed is markedly acetonitrile with a content of 10% propionitrile.

is borrowed. The temperature for the 8 ml (0.087 mol) phosphorus oxychloride according to the invention, 1 ml (0.056 mol)

Procedure should be in conjunction with the amount of water and 8 ml (0.082 mol) of «picoline, have been used at

organic base can be determined, but it is 0 to 5 ^ C 5.4 g (0.022 mol) inosine with stirring.

give; the reaction was continued for 5 hours. Following the procedure of Example 1, 7.1 g of 97% purity sodium inosinate was obtained.

Example 4

To a mixed solution containing 60 ml of nitromine 24 ml (0.262 moles) of phosphorus oxychloride. 2.6 ml (0.144 mol) water and 23.7 ml (0.294 mol) pyridine. kept at about 5 ° C, 17 g (0.060 mol) of guanosine was added with stirring. After the reaction was allowed to run for 5 hours. the reaction mixture was poured into about 2 liters of cold water poured and stirred for about 1.5 hours. Then it was left standing.

The resulting aqueous solution containing 2% guanosine, 1% guanine, 92% guanosine-5'-monophosphate and Containing 5% other phosphates was passed through a column containing 150 g of activated carbon, and then it was washed with water. The column was then filled with 2.5 liters of 0.25 η-aqueous sodium hydroxide eluted. The eluate was then passed through a column with about 50 ml of an ion exchanger run in the Η-shape; the column was washed with water. The draining liquid and the washes were combined, then sodium hydroxide was used to adjust the pH to the resulting pH Mix adjusted to 7.6; the mixture was concentrated under reduced pressure, whereby about 100 ml of concentrated solution were obtained. The resulting concentrated solution was 300 ml Methanol added; they were left in a refrigerator overnight. The crystals formed were filtered off and dried; 20.6 g of disodium guanosine 5'-monophosphate were obtained, containing 97% 5'-guanylate and 1% other phosphates.

Example 5

To a mixed solution consisting of 60 ml of acetonitrile, 24 ml (0.262 mol) of phosphorus oxychloride. 2.6 ml (0.144 mol) of water and 24 ml of a mixture containing η-, β-, / -picoline and 2,6-lutidine in a ratio of 7: 1: 1: 1 (about 0.238 mol of picoline and lutidine) kept at 0 to 2 ° C, 17 g (0.060 mol) of guanosine was added and the reaction was allowed to proceed for about 6 hours. Following the procedure of Example 4, 19.5 g of sodium guanylate containing 96% guanosine 5'-monophosphate and 2% other phosphates were obtained.

Example 6

Example 4 was repeated with the difference that acetonitrile and 16 g (0.061 mol) of adenosine were used instead of nitromethane or guanosine. As a result, there was 20 g of sodium adenylate obtained with a content of 95% adenosine S'-monophosphate and 2% other by-products.

_{| 0} B eis ρ ie I 7

To a mixed solution containing 20 ml of dichloromethane, 10 ml (0.109 mol) of phosphorus oxychloride, 1.2 ml (0.067 mol) of water, 9.5 ml (0.117 mol) of pyridine kept at 5 to TC was added 5.4 g (0.022 mole) inosine was added with stirring and the reaction allowed to run for 4 hours. Working according to Example 1, 6.8 g of sodium inosinate with a content of 90% 5'-monophosphate and 3% other phosphates were obtained.

Example 8

Example 1 was repeated, but using 5.7 g (0.020 mol) of xanthosine instead of inosine became. 6.6 g of pure sodium xanthosine S'-monophosphate were obtained.

Example 9

To e · item mixed solution containing 20 ml acetonitrile, 8 ml (0.087 mol) of phosphorus oxychloride and 7.9 ml (0.098 mol) of pyridine, which was kept at 0 to 2 "C, 5.8 g (0.019 mol) of inoskidihydrate (0.038 mol of crystalline water) were added. When working according to the example 1, 6.0 g of 5'-inosinate with a purity of 90% were obtained.

Example 10

6 ml (0.66 mol) of phosphorus oxychloride were added to 15 ml of acetonitrile. 0.72 ml (0.040 mol) of water and 7.2 ml (0.074 mol) of n-picoline were added. To the resulting To the mixture kept at 0 to 2 ° C, 3.7 g (0.015 mol) of cytidine was added with stirring. When working according to Example 1, 5.0 g of disodium cytidine-5'-phosphate were obtained in 99% purity

. receive.

Example 11

Example 10 was repeated, but using 3.7 g (0.015 mol) of uridine instead of cytidine became. 5.1 g of disodium uridirl 5'-phosphate were obtained in 97% purity.

409640/60

Claims (1)

64i979 Patent claims: I. Process for preparing ribonucleoside-5-monophosphate of the general formula HO O \ ll PO-CH ₂ , 0 HO OH OH in which B denotes the residue of a purine or a pyrimidine base, by selective phosphorylation of the 5'-hydroxyl group of the corresponding ribonucleoside unprotected in the T- and 3'-positions, characterized in that a ribonucleoside of the general formula HO-CH ₂ OB HI IH OH OH